Ischemia-hypoxia followed by reperfusion and reoxygenation injures cells and organs. Previous studies have indicated that isoflurane may protect organs from ischemia-reperfusion or hypoxia-reoxygenation. This study investigated the ability of isoflurane to protect the liver from hypoxia-reoxygenation injury and the mechanisms of this phenomenon.
The isolated liver was perfused at a constant pressure of 12 cm H2O with a modified Krebs-Ringer-bicarbonate solution saturated with a 95% oxygen/5% carbon dioxide gas mixture. Hypoxic perfusion produced by decreasing the oxygen concentration in the gas mixture to 10% was followed by perfusion at 95% oxygen for 60 min. Viability of the liver was assessed by lactate dehydrogenase release from the liver. Isoflurane at 0.5, 1, and 2 minimum alveolar concentration was administered to assess the effect of isoflurane on hypoxia-reperfusion injury. To determine the effect of isoflurane on extracellular generation of superoxide in the liver, the reduction of ferricytochrome c with or without superoxide dismutase was measured.
Lactate dehydrogenase release was transiently but dramatically increased by reoxygenation and significantly attenuated by 1 and 2 minimum alveolar concentration of isoflurane. Suppression of Kupffer cells with gadolinium chloride also attenuated the lactate dehydrogenase release. Isoflurane significantly reduced the superoxide generation on reperfusion.
The results show that isoflurane protected the liver from an early reoxygenation injury presumably mediated by Kupffer cells. The mechanisms of the inhibitory effects of isoflurane on the injury may involve suppression of extracellular superoxide generation during reoxygenation.
Ischemia/hypoxia followed by reperfusion-reoxygenation causes cell and organ injury. Ischemia-reperfusion or hypoxia-reoxygenation injury may be involved in hepatic dysfunction, liver transplantation, hemorrhagic and septic shock, and in hepatic surgery requiring repeated cross-clamping of the portal vein and hepatic vascular exclusion. Although intraoperative ischemia-reperfusion or hypoxia-reoxygenation of the liver generally occurs under general anesthesia, little is known about the direct effect of anesthetic agents on hepatic injury due to this phenomenon. Previous studies have suggested that isoflurane may protect organs from ischemia-reperfusion or hypoxia-reoxygenation. For example, isoflurane partly prevents a reduction in both energy charge and total adenine nucleotide in isolated hepatocytes during anoxic challenge, [4,5]and it protects the brain during hypoxia or ischemia. Recent investigations from our laboratory have indicated that isoflurane may protect the isolated perfused liver from early, neutrophil-independent, ischemia-reperfusion injury by acting mainly during the reperfusion phase. 
Isolated liver perfusion has been used to explore the mechanisms of reperfusion injury, because this technique can control precisely the various physiologic factors that affect the injury. Unlike isolated hepatocytes, the isolated perfused liver preserves the interaction of parenchymal and nonparenchymal liver cells, which has been shown to be involved in ischemia-reperfusion injury. [9,10]In our study, we determined the effects of hypoxic duration on the injury, of isoflurane on the hypoxia-reoxygenation injury, of suppression of Kupffer cells on the reoxygenation injury, and of isoflurane on superoxide generation during reoxygenation in the isolated perfused liver.
Materials and Methods
The experiments were approved by the Animal Investigation Committee of Chiba University School of Medicine and were performed according to our institution's Guide for Animal Experimentation.
Animals and Care
Eight-week-old, male Sprague-Dawley rats (body weight, 250–310 g) were purchased from Nihon SLC (Shizuoka, Japan). The animals were acclimated to the environment of the animal research facility for at least 1 week. The room temperature was kept at 24–26 degrees Celsius. The animals had free access to a standard laboratory chow and water under a 12:12 h light-dark cycle. All animals were fasted for 24–30 h before the experiment.
The perfusion medium used was a modified Krebs-Ringer-bicarbonate (mKRB) solution. It contained 117 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl2, 1.19 mM KH2PO4, 1.44 mM MgSO4, and 24.8 mM NaHCO3. The mKRB solution was saturated with a 95% oxygen-5% carbon dioxide gas mixture, and pH was adjusted to 7.35 +/- 0.05 at 37 degrees Celsius using 1 N NaOH or NaHCO3, (or both).
Isolated Perfusion of the Rat Liver
Each liver was excised and perfused as described previously. Briefly, each rat was anesthetized with an intravenous injection of pentobarbital sodium (35 mg/kg) through the dorsal penile vein, and its abdomen was exposed. Heparin sodium was intravenously injected at 50 units, and then the abdominal inferior vena cava was ligated above the left renal vein. A polyethylene catheter (PE-240) was inserted into the portal vein and secured in place. A non-recirculating perfusion through the portal vein with mKRB was initiated immediately and continued during the following surgical procedure to minimize hypoxia. After exposure of the thorax, another polyethylene catheter (PE-260) was inserted through the right atrium and secured in the thoracic inferior vena cava. The liver was gently excised and placed in a modified Miller-type recirculating perfusion-aeration chamber. In this system, the liver was perfused at a constant pressure of 12 cm H2O with mKRB containing 1% bovine serum albumin and 10 mM D(+) glucose. Hepatic flow was monitored continuously using an electromagnetic flow transducer (Nihon Koden, Tokyo, Japan). Two needle-type oxygen electrodes (Intermedical, Nagoya, Japan) were placed in both inflow and outflow chambers to monitor partial pressure of oxygen (PO2). The liver was perfused initially at 95% oxygen. The perfusate temperature was kept automatically at 37 degrees Celsius, and pH was maintained in a range of 7.35 +/- 0.05 by continuous infusion of 1 N NaOH.
Effects of the Duration of Hypoxia on Hypoxia-Reoxygenation Injury
The hepatic flow and oxygen consumption were stabilized 20 to 30 min after the initiation of recirculating perfusion. After 20 min of equilibration (time = 0 min), hypoxic perfusion at 10% oxygen was initiated in the hypoxic group. Hypoxia was maintained for 15, 30, 45, 60, or 90 min and followed by reoxygenation at 95% oxygen for 60 min. In the control group, the liver was perfused at 95% oxygen throughout the experiment.
Effects of Isoflurane on Basal Hepatic Flow, Carbohydrate Metabolism, and Lactate Dehydrogenase Release
Immediately after hepatic flow and oxygen consumption were stabilized (time =-20 min), the livers in the isoflurane groups were exposed to isoflurane at a concentration equivalent to 2 minimum alveolar concentration (MAC; 2.9% in rats), 1 MAC (1.5%), or 0.5 MAC (0.7%). Isoflurane was administered until the end of the experiment (time = 120 min). The control livers were not exposed to isoflurane.
Effects of Isoflurane on Hypoxia-Reoxygenation Injury
Immediately after hepatic flow and oxygen consumption were stabilized (time =-20 min), the liver in the isoflurane group was exposed to isoflurane at 2, 1, or 0.5 MAC. Isoflurane was administered continuously throughout the experiment. Hypoxic perfusion at 10% oxygen was initiated in all hypoxic groups after 20 min of equilibration (time = 0 min). Hypoxic perfusion was maintained for 60 min, followed by reoxygenation at 95% oxygen. Reoxygenation persisted for 60 min.
Effects of Suppression of Kupffer Cells on Hypoxia-Reoxygenation Injury
To study whether suppression of Kupffer cells affects the hypoxia-reoxygenation injury, gadolinium chloride (GdCl3), which suppresses and destroys Kupffer cells selectively, [9,14]was given intravenously (7.5 mg/kg) through the dorsal penile vein 24–30 h before liver perfusion. This dose and timing of administration exerts a maximal effect on Kupffer cells. Control rats received the same volume of 0.9% saline. Isoflurane administration and hypoxic challenge followed by reoxygenation reoxygenation were performed as previously described.
Effects of Isoflurane on Superoxide Generation Immediately after Reoxygenation
Previous studies have shown that a transient burst of oxygen radical occurs immediately after reoxygenation in the isolated liver subjected to hypoxia or ischemia. [15,16]To determine the effect of 2 MAC isoflurane on superoxide generation that occurs immediately after reoxygenation, we determined the activity of superoxide release by reducing ferricytochrome c in the following groups:(1) a control group without hypoxic challenge or isoflurane administration, (2) an isoflurane-treated group with no hypoxic challenge, (3) a control group with a hypoxic challenge, and (4) an isoflurane-treated group with a hypoxic challenge. The reduction of cytochrome c is not inhibited by isoflurane. The mKRB solution containing 10 mM glucose and 80 micro Meter ferricytochrome c (from horse heart; Sigma Chemical Co., St. Louis, MO) was saturated with a 95% oxygen-5% carbon dioxide gas mixture in the control groups. The solution was saturated with a 95% oxygen-5% carbon dioxide gas mixture and 2 MAC isoflurane in the isoflurane-treated groups. The hypoxic challenge and isoflurane administration were performed as described previously. At 60 min, the mKRB solution containing ferricytochrome c was perfused to the liver at a flow rate of 50 ml/min in a non-recirculating manner. During 30–60 s of the non-recirculating perfusion, the perfusate was collected from the outflow chamber and immediately placed in an ice bath. Within a few minutes, the content of reduced ferricytochrome c was determined as the change in absorbance at 550 nm using a U best-35 type spectrophotometer (Nihon-Bunkoh). The reduction of ferricytochrome c is not an assay specific for superoxide. The required specificity is achieved using superoxide dismutase (SOD; Sigma Chemical Co.), for which superoxide is the only known substrate. Accordingly, the assay using the mKRB solution containing 80 micro Meter ferricytochrome c and 10 micro gram/ml SOD were also performed in the four treatment groups.
Forawik (Murako, Tokyo, Japan) was used for isoflurane vaporization. The flow rate of the oxygen-carbon dioxide mixture into the perfusion system was set at 21 l/min. The accuracy of the vaporizer at this gas flow rate was checked with a Capnomac anesthetic gas analyzer (Datex, Helsinki, Finland). The concentration of isoflurane was determined in preliminary experiments by a gas chromatographic head-space analyzer. Isoflurane was equilibrated in the solution within 15 min of the initial administration (Table 1).
Measurement of Variables
Oxygen consumption of the perfused liver was calculated by the following equation:Equation 1where a is Bohr's coefficient (24 ml oxygen/ml water at 760 mmHg at 37 degrees Celsius). Approximately 1 ml perfusate was collected sequentially. The perfusate concentrations of D-glucose and L-lactate were determined immediately using a YSI 2300 analyzer (YSI, Yellow Springs, MI). Approximately 900 micro liter perfusate was stored at -20 degrees Celsius in a freezer until the lactate dehydrogenase (LDH) assay. Lactate dehydrogenase activity was determined at 37 degrees Celsius with a Hitachi 736 autoanalyzer (Tokyo, Japan). Isoflurane did not interfere with the LDH assay. Net glucose and lactate productions (mM/min) and LDH release (IU/min) were computed using a pair of determined values.
Repeated-measures analysis of variance was performed for serial measurements to assess change over time and the effect of treatments (isoflurane concentration or hypoxic challenge or both). A multivariate test based on the Rao R criteria was applied to explore the interaction between treatment and time. Between-group and within-group comparisons were made using contrast. One-way analysis of variance followed by Scheffe's F test was performed for nonserial measurements. These statistical analyses were performed using a statistical software package (Statistical 5.0, Statsoft, Tulsa, OK). The difference was considered significant when the probability value was less than 0.05. All values were expressed as mean +/- SD.
Effects of the Duration of Hypoxia on Reoxygenation Injury
Because the duration of perfusion may influence LDH release, we compared the level of released enzyme between one control group and each hypoxic group on an absolute time scale. As shown in Table 2, the hepatic injury assessed by LDH release became significant when the duration of hypoxia was 60 and 90 min. Lactate dehydrogenase release during hypoxia was moderately augmented by 60-min hypoxia, whereas it was considerably increased by 90-min hypoxia. Thus the liver was severely damaged during hypoxia when it lasted 90 min. Release of LDH markedly increased during the initial 0–15 min of reoxygenation when hypoxia lasted 60 and 90 min. Lactate dehydrogenase release after 15 min of reoxygenation did not significantly increase even when hypoxia lasted 90 min. Because hypoxic perfusion for 60 min was sufficient to produce reoxygenation injury, we used it to study the effects of isoflurane.
Effects of Isoflurane on Basal Hepatic Flow, Metabolism, and Lactate Dehydrogenase Release
As shown in Figure 1(A), isoflurane maintained basal hepatic flow. Isoflurane at 1 and 2 MAC decreased basal oxygen consumption. There were no significant differences in basal glucose production among the treatment groups. The effect of isoflurane on basal lactate production depended significantly on time. Isoflurane at 1 and 2 MAC caused a brief increase in lactate production at time = 0 min, whereas it attenuated lactate production after time = 60 min (Figure 2(A)).
(Figure 3(A)) shows the changes in LDH release in the perfusion medium. Release of LDH remained low until time = 60 min, but it increased slightly after time = 75 min. There were no significant differences among groups.
Effects of Isoflurane on Hypoxic-Reoxygenation Injury
As shown in Figure 1(B), hypoxic perfusion at 10% oxygen produced a large decrease in hepatic oxygen consumption in all animals. Reoxygenation restored hepatic oxygen consumption in all treatment groups. Although hepatic flow and oxygen consumption gradually decreased during reoxygenation in all groups, hepatic flow during reoxygenation was kept higher in the 1 and 2 MAC isoflurane-treated groups than in the control group.
As shown in Figure 2(B), the net lactate production increased before the hypoxic challenge in the isoflurane groups. Hypoxic challenge moderately increased net glucose and lactate productions. Isoflurane did not significantly modulate these hypoxic changes. Reoxygenation produced transient but dramatic changes in net glucose and lactate productions. Lactate production sharply decreased and became negative, indicating that reoxygenation initially stimulated lactate uptake. Glucose production increased temporally after reoxygenation. Lactate production gradually increased and glucose production diminished during the later reoxygenation period. Isoflurane at 1 and 2 MAC significantly modified these changes. It suppressed the reoxygenation-induced lactate uptake.
A moderate increase in LDH release was observed during hypoxia. The release of LDH greatly increased during the initial 15 min of reoxygenation (Figure 3(B)). The release of this enzyme was significantly less in the 1 and 2 MAC isoflurane groups compared with the control group.
Effects of Suppression of Kupffer Cells on Hypoxia-Reoxygenation Injury
As shown in Figure 4, the release of LDH was significantly less in the isoflurane- and gadolinium-treated groups compared with the control group.
Effects of Isoflurane on Superoxide Generation Immediately after Reoxygenation
(Figure 5) shows the difference in absorbance at 550 nm between perfused and nonperfused solution. With no SOD in the solution, the difference was significantly larger in the control group subjected to hypoxic challenge than in any of the other three groups. The difference in absorbance did not differ significantly between the two isoflurane-treated groups with and without hypoxia. In the presence of SOD in the solution, there were no significant differences in the absorbance difference among the treatment groups. These results indicate that superoxide generation immediately after reoxygenation was inhibited significantly by 2 MAC isoflurane.
The primary finding of this study is that 1 and 2 MAC isoflurane protected hepatic cells from injury during the early phase of hypoxia-reoxygenation, as shown by a reduction in reoxygenation-induced increase in LDH release. Our results, which correspond with those of previous reports, [9,20]also indicate that suppression of Kupffer cells with GdCl3reduced the hypoxia-reoxygenation injury. Furthermore, isoflurane suppressed the extracellular generation of superoxide after reoxygenation. The activity of LDH in the perfusate was an indicator of hepatocellular damage. This is based on the previous report that LDH release from the liver corresponds to trypan blue labeling of hepatocellular nuclei, an established indicator of cell death. 
It is generally believed that reactive oxygen is involved in the reperfusion injury. Results of several studies using xanthine oxidase inhibitors, reactive oxygen scavengers, superoxide dismutase, and catalase have supported the hypothesis of Granger and associates that reactive oxygen generated in parenchymal cells by cytosolic xanthine oxidase can cause reperfusion injury. [23–25]However, sufficient evidence has accumulated recently showing that intracellular oxidant stress can be detoxified in the hepatocytes, which have a high antioxidant capacity. In addition, intracellular reactive oxygen formation during the reperfusion period may be insufficient to cause hepatocellular injury. [20,26–28]Experiments with the isolated, blood-free perfused liver have suggested that extracellular oxidant stress, derived presumably from activated Kupffer cells, may also cause the initial vascular and parenchymal cell injury. [10,28,29]
Previous studies have shown that a transient burst of oxygen radical occurs immediately after reoxygenation in the isolated liver subjected to hypoxia or ischemia. [15,16]In our study, extracellular generation of superoxide after reoxygenation was determined using the reduction of ferricytochrome c in the presence and absence of SOD. In the absence of SOD, hypoxia significantly augmented the reduction in the control groups but not in the isoflurane-treated groups. With SOD, hypoxia did not significantly augment the reduction of ferricytochrome c in either the control or isoflurane-treated groups. These results suggest that reoxygenation after 60 min of hypoxia augments the generation of superoxide and that the mechanism of inhibitory action of isoflurane on hypoxia-reoxygenation injury may involve the inhibition of extracellular superoxide generation on reperfusion.
Previous studies show that isoflurane may attenuate the superoxide generation of Kupffer cells on reperfusion. Araki and coworkers report that isoflurane may elicit beneficial effects on the liver by attenuating the protein kinase C-mediated alterations in hepatic hemodynamics and metabolism. Furthermore, protein kinase C is inhibited by alcohol and anesthetics. Protein kinase C is involved in activating the reduced nicotinamide adenine dinucleotide phosphate oxidase, which stimulates extracellular superoxide production. Therefore, one possible mechanism of isoflurane-induced reduction of superoxide generation may be that isoflurane affects the activation of the reduced nicotinamide adenine dinucleotide phosphate oxidase by attenuating protein kinase C-mediated alterations.
Another possible explanation for the protective effect of isoflurane on hypoxia-reoxygenation injury is that isoflurane may suppress metabolism or depress adenosine triphosphate utilization. In accord with our previous studies, [7,30]the present study (Figure 1) showed that isoflurane decreased oxygen consumption. Although the mechanism involved in the decrease of oxygen consumption by isoflurane is not fully clear, isoflurane suppresses various energy-requiring processes in the liver. [4,5]When the oxygen supply to the liver is diminished, suppression of oxygen consumption by isoflurane may become beneficial.
Furthermore, the mechanism of action of isoflurane on hypoxia-reoxygenation injury may involve the modulation of intracellular pH. Intracellular acidosis increases the viability of isolated hepatocytes and perfused liver during hypoxia followed by reoxygenation. The medium pH was maintained within the physiologic range (7.30–7.40) in this study. However, isoflurane might modify intracellular pH by affecting carbohydrate metabolism, because it enhanced net lactate production before hypoxia. In addition, a previous report suggested that lactate accumulation during ischemia may be beneficial and that supplementation with lactate could protect against postischemic injury. 
For this study, we selected isoflurane as a volatile anesthetic. It would be interesting to know whether other volatile anesthetics have a similar protective action. Recently we tested the effects of halothane, isoflurane, and sevoflurane on ischemia-reperfusion injury in the isolated perfused liver. We induced ischemia by decreasing the perfusion pressure from 12 to 2 cm H2O. With that model, all volatile anesthetics attenuated the ischemia-reperfusion injury, and isoflurane had the most prominent effect.
In the present study, we tested the effect of isoflurane on the isolated perfused liver subjected to 60 min of hypoxia. Preventing reperfusion injury after an extensive ischemic injury has no measurable effect on organ function. We selected this hypoxic period because it produce a negligible hypoxic injury but an obvious reoxygenation injury that occurs during the initial 15 min of reoxygenation. In subsequent experiments, reoxygenation was maintained for 60 min. Thus our model focused on the effect of isoflurane on early reoxygenation injury mediated predominantly by Kupffer cells.
However, our model is clearly different from other in vivo ischemia-hypoxia and reperfusion models. Our model excluded extrahepatic factors that may influence the injury process. The liver in this model is not influenced by blood components, particularly neutrophils. The absence of neutrophils in the perfusate may limit the significance of our results, because accumulation of neutrophils has been demonstrated in various reperfused organs, including the liver. Activated neutrophils can produce reactive oxygen, a major mediator of the reperfusion injury. However, evidence has accumulated that neutrophils are not involved in the early phase of reperfusion injury in the liver. Thus the reoxygenation injury observed during the initial 15-min reoxygenation period in the present study was consistent with the early reoxygenation injury that is independent of neutrophils.
Our results from the isolated perfused liver cannot be applied clinically. The effect of isoflurane on systemic hemodynamics was not considered in our model. Isoflurane may reduce cardiac output and alter its distribution to the splanchnic circulation. [36,37]However, these effects may be minimized by proper fluid therapy, blood transfusion, or the administration of inotropic agents. Our model did not allow assessment of the effects of isoflurane on neutrophil-dependent, late-reperfusion injury reported to occur 6–24 h after reperfusion. However, activation of Kupffer cells in the early reperfusion probably primes the following neutrophil-dependent injury. [38,39]Furthermore, isoflurane suppresses superoxide generation in neutrophils. Thus isoflurane may attenuate the neutrophil-dependent, late-reperfusion injury.
Using an isolated, blood-free perfused liver, we found that isoflurane protected the fasted liver from an early, neutrophil-independent reoxygenation injury. The mechanisms of action of isoflurane in hypoxia-reoxgenation injury may involve the reduction of superoxide generation on reoxygenation.